Immobilization of Cholesterol Oxidase in Chitosan Magnetite Material for Biosensor Application

Cholesterol oxidase, a bio-catalyst that can catabolize cholesterol, has proven applications in medicine. Here, a support material was used to enhance the characteristics of the enzyme. Magnetite (Fe₃O₄) is widely used as an enzyme support; however, the interaction between the enzyme and the support should be capped with another material, such as chitosan biopolymer-based material. In this study, chitosan-magnetite materials were synthesized by mixing both compounds and activating with glutaraldehyde. The materials were then characterized by Fourier Transform Infrared (FTIR) Spectroscopy. The enzyme kinetic parameters were studied by following the cholesterol oxidation reaction using high-performance liquid chromatography (HPLC) and comparing the results between the free and the immobilized enzyme. The substrate concentration was 2.5 mg/mL. The effect of enzyme concentration was tested using different concentrations of enzyme (0.5, 1, and 2 mg/mL) to determine the best operating conditions. The best conditions for the oxidation reaction were immobilized enzyme at a 2 mg/mL concentration. Enzyme immobilization significantly decreased the optimum substrate concentration to 0.1 mg/mL.

Cholesterol; Cholesterol oxidase; Immobilized enzyme; Magnetite; Oxidation

The enzymatic method is more advantageous, as it is not corrosive and is run as a specific reaction. However, the enzymatic method still has disadvantages that include enzyme inactivation under abnormal conditions of temperature and pH. The specific enzyme used for enzymatic cholesterol is cholesterol oxidase. This enzyme is produced by several pathogenic and non-pathogenic microorganisms, such as Mycobacterium, Brevibacterium, Streptomyces, Corynebacterium, Arthrobacter, Pseudomonas, Rhodococcus, Chromobacterium, and Bacillus species. Cholesterol oxidase from Streptomyces sp. can also reportedly oxidize the substrate from fatty foods and can degrade up to 80% of the initial concentration of the substrate (Perdani et al., 2019a).

Cholesterol oxidase kinetic behavior has been investigated with a first order irreversible reaction model. The enzymatic reaction needs one or two enzymes to break the structure into a complex component (Perdani et al., 2019b). The enzyme acts as an electron donor to the CH-OH group in cholesterol. The final cholesterol enzymatic oxidation product is 5-cholesten-3-one, which is isomerized through three stages to produce 4-cholesten-3-one (Devi and Kanwar, 2017). In the first catalytic stage, dehydrogenation of the OH group results in the loss of two hydrogen molecules in the third steroid ring. The released hydrogen molecules are transferred to the FAD enzyme cofactor so that FAD is reduced. The reduced FAD cofactor then reacts with oxygen molecules to restore the initial condition of the enzyme, where the FAD is re-oxidized and the hydrogen atom reacts to form H₂O₂. The final stage of this process is the isomerization of the double-chain steroid ring and production of the final product, 4-cholesten-3-one (Devi and Kanwar, 2017).

Enzymatic methods have been applied in many research applications, such as renewable energy, bio-products, and pharmaceuticals. Enzymes can have multiple functions and can be used as catalysts, extraction agents, and capping agents. The process parameters of temperature, enzyme substrate concentration, and extraction time have the greatest effects on the enzymatic reaction. In addition, the reaction slows down unless enzyme is continuously added (Handayani et al., 2018). However, a study of enzymes used as biocatalysts to produce biodiesel has shown that immobilization of the lipase enzyme by an adsorption-crosslinking method stabilized the enzyme and kept it soluble in the reaction. Assays of the immobilized enzyme in the biodiesel synthesis reaction revealed that it retained 84% of its initial activity (Aliyah et al., 2016). Similarly, Hermansyah et al. (2018), examined a lipase enzyme from Pseudomonas aeruginosa by a fermentation method for biodiesel production from palm oil mill effluent (Hermansyah et al., 2018). Therefore, immobilization is a commonly used method to improve enzyme activity.

Enzyme immobilization requires a material that will act as a support to stabilize the enzyme. Various kinds of supports are used for immobilization, including polymers, carbon, metals, or metal-metal combinations. One common support used in enzyme immobilization is chitosan. Chitosan is a biopolymer with a number of advantages; it is renewable, nontoxic, and highly available in Nature (Peter, 1995). In industrial applications, chitosan is a source for composites of activated clinoptilote zeolite/chitosan used as a support for biogas purification (Kusrini et al., 2019). According to Ahmad and Goswami (2014), chitosan also can be used as a support for bioreactions. It can improve the characteristics of the cholesterol oxidase enzyme against changes in reaction temperature. For example, the activity of an immobilized enzyme can be maintained up to 50°C, where it can still show 77% activity after 12 repeated enzyme reaction cycles (Ahmad and Goswami, 2014).

Chitosan and agarose are the most common biopolymers used as enzyme immobilization supports. Biopolymers used as immobilization supports bind the enzyme by adsorption and covalent bonding as immobilization techniques. However, the ability of biopolymers to form geometric structures, such as gel forms, makes them also useful for immobilization techniques involving encapsulation and entrapment (Zdarta et al., 2018).

In addition to biopolymers, inorganic and organic materials are also widely used in the preparation of enzymes, due to their specific characteristics. Magnetite particles have been attracting much interest for the development of many applications because of their unique properties, such as small size, superparamagnetism, low toxicity, good biocompatibility, and high surface area. Previous studies have also confirmed that bioenzymes can be immobilized with magnetic chitosan. The ability to undergo covalent binding produced strong enzyme activity and immobilization was confirmed by characterization of the sample with FTIR (Hamzah et al., 2019).

Magnetite nanoparticles dispersed in chitosan have been used as an electrochemical detector for the determination of the endocrine disruptor parathion. This composite showed good detection of the specific target compound. Chitosan has also been used to improve the stability of magnetite and to functionalize the surface of particles. The performance of a detector was improved with an electrode modified with magnetite and chitosan when compared with an unmodified electrode. The adsorption of magnetite was also increased after the addition of chitosan, as the matrix components of chitosan had a great influence on magnetite (Piovesan et al., 2018).

Magnetite-chitosan materials are widely used because of their easy separation and great support. A lipase enzyme covalently immobilized with magnetite chitosan was found to maintain 75.5% of its initial activity for 6 h. The magnetite chitosan has a great influence on the specific properties of enzyme, as it allows the creation of monolayers composed of different types of lipids, sterols, and their mixtures (Suo et al., 2018). In the present research, magnetite chitosan has been used as a support for cholesterol oxidase.

Magnetite chitosan has also been used for non-enzymatic reactions, but non-enzymatic reactions are slower than enzymatic methods. The aim of the present study was to explore the use of modified magnetite chitosan for the enzymatic reaction of cholesterol oxidation. A further aim was to investigate the effect of enzyme immobilization on the oxidation reaction between immobilized cholesterol oxidase and its substrate. The enzyme was tested for its ability to conduct the oxidation reaction by varying the enzyme concentration, substrate concentration, and reaction time. The immobilized enzyme material was characterized by Fourier transform infrared (FTIR) spectroscopy and the oxidation reaction was quantified by high performance liquid chromatography (HPLC). 

    Cholesterol oxidase immobilized with chitosan magnetite was able to oxidize up to 90% of a cholesterol substrate at different reaction times. The enzyme interacted with magnetite nanoparticles covered with aminated chitosan. The NH₂ functional group was recorded during the immobilization step. Cholesterol oxidation with the immobilized enzyme showed that the use of a support material can significantly change the behavior of the enzyme to oxidize the substrate. However, the concentration of enzyme also affected the behavior of the oxidation reaction. Chitosan-magnetite could be a candidate for a cholesterol biosensor due to the sensitivity of the oxidation reaction for the substrate. The chemical properties are better for the immobilized enzyme than for the free enzyme. The best concentration of the immobilized enzyme for substrate oxidation was 2 mg/mL with the maximum reaction time.